Molecular analysis

ABSTRACT

A spectrometer for analysing material comprises a light source, a monochromator for selecting a range of wave-lengths from the light source and emitting them as monochromatic light, a chamber for locating a sample, a focusing means for focusing the monochromatic light onto a sample in the chamber, a detector for measuring the monochromatic light after it has interacted with the sample. An independently variable parameter is varied between two values vi and v2, while the detector measures the monochromatic light across a range of is wavelengths, the independent variable having a value or values between v1 and v1+Δv, and Δv being much smaller than the interval between v1 and v2.

This invention relates to molecular analysis, using one or morespectroscopic probes, particularly a circular dichroism spectroscopicprobe.

When a sample containing a chiral chromophore is alternately radiated byleft circularly polarised left and right circularly polarised light, theleft circularly polarised light will be absorbed to a different extentthan the right circularly polarised light. Measuring the difference inabsorption ΔA between the left and right circularly polarised light as afunction of wavelength gives a circular dichroism spectrum which cangive information about the sample, for instance the structure of aprotein. Of particular interest is how the structure of the proteinchanges with temperature, since the integrity of a protein's secondarystructure gives a good indication of how stable it will be in solution.In this case, equal amounts of left and right circularly polarised lightof a particular wavelength are directed at a sample and the temperatureis changed continuously during the measurement. In this way,temperature-induced change in the protein secondary structure may beobserved. However, choosing an appropriate wavelength for themeasurement assumes a-priori knowledge of the protein, which is notalways the case.

Alternatively, one can take a series of measurements on discrete samplesfor a sequence of wavelengths; but for irreversible denaturation (quiteusual), measuring sequentially at more than one wavelength requires anew sample of the protein for each experiment and it may be in shortsupply. Then, the interpretation of the data is problematic because itis likely to be dependent on a very limited subset of the possible data.The complete measurement process for a sample is also relatively slowcompared to other techniques.

The object of the present invention is to provide a fast and convenientmethod of obtaining and analysing circular dichroism spectra.

Accordingly to the present invention there is provided a spectrometerfor analysing material comprising

a light source

a monochromator for selecting a range of wavelengths from the lightsource and emitting them as monochromatic light

a chamber for locating a sample

a focusing means for focusing the monochromatic light onto a is samplein the chamber

a detector for measuring the monochromatic light after it has interactedwith the sample

wherein an independently variable parameter is varied between two valuesv1 and v2

and the detector measures the monochromatic light across a range ofwavelengths when the independent variable has a value or values betweenv1 and v1+Δv, where Δv is much smaller than the interval between v1 andv2.

According to another aspect of the present invention, there is provideda spectrometer for analysing material comprising

a light source

a monochromator for selecting a range of wavelengths from the lightsource and emitting them as monochromatic light

a chamber for locating a sample

a focusing means for focusing the monochromatic light onto a sample inthe chamber

a detector for measuring the monochromatic light after it has interactedwith the sample

wherein the detector is an avalanche photodiode detector.

Recording the CD spectra in this way combines the desirablecharacteristics of speed and multiple wavelengths and assumes noa-priori knowledge of the protein. It is likely that more data pointscan be measured, which makes the process of analysing the proteinstructure with temperature change more accurate and precise.

In particular it makes the calculation of melting points and enthalpiesof is transitions very much faster and more robust; it confirms that theprotein is correctly folded initially; it identifies the components ofthe secondary structure that change; and it differentiates betweenunfolding (where a protein molecule changes its three-dimensionalstructure) and aggregation (where protein molecules come together inclumps). This allows an estimation of the protein's stability to bemade, a confirmation that the sample is the correct, active, protein,and the unfolding behaviour gives chemists guidance as to formulation.

The use of the Wollaston prism arrangement in the monochromator, givingimproved light bandwidth, and the use of an avalanche photodiodedetector, giving improved signal-to-noise characteristics, both helpimprove the rate at which readings can be taken across a range ofwavelengths while the sample has a particular variable held constant orallowed to move through a small interval, so that the spectrometer canassess a useful number of samples in a practical time scale.

The invention will now be described, by way of example, and withreference to the accompanying drawings, of which:

FIG. 1 is a schematic view of the lamp housing;

FIG. 2 is a schematic view of the monochromator;

FIG. 3 is a schematic view of the light conditioning unit and the samplechamber;

FIG. 4 is a graphical representation of circular dichroism spectra of isa sample;

FIG. 5 is a graphical representation of the difference in circulardichroism vs temperature of a sample;

FIG. 6 is a graphical representation of the light absorbance vstemperature of a sample;

FIG. 7 is a graphical representation of the circular dichroism spectraof the independent species of a sample;

FIG. 8 is a graphical representation of the concentration of theindependent species as a function of temperature;

FIG. 9 is a graphical representation of the transition surface for thethree independent species together as a function of temperaturewavelength and CD difference;

FIG. 10 is a graphical representation of the transition surface for theresidue together as a function of temperature wavelength and CDdifference.

Referring to FIG. 1, white light (whose path is generally indicated by amiddle line and two outer lines 20) is produced from an intense lightsource 10 chosen to have a good output throughout the ultra-violetregion of the spectrum down to approximately 180 nm, such as a xenon arclamp with a pure silica envelope. This light is focused by a concavereflector, such as an ellipsoidal mirror, 12 through a aperture 14 intothe entrance 16 to a is polarising monochromator.

Referring to FIG. 2, the light 20 passes through the entrance slit 16 ofthe polarising monochromator and falls on a mirror 22 which reflects thelight onto a first prism 24. The prism disperses and polarises the lightso that diverging ordinary and extraordinary beams of linearly polarisedlight comprising a limited band of wavelengths fall on a second mirror26. (For clarity, only one polarisation state is shown post-mirror 26.)Part of the beams pass through the slit 27, which selects for wavelength(because the light is dispersed) and polarisation state (because thepolarised beams are divergent). The selection process means that arelatively monochromatic ordinary beam centred about one wavelength anda relatively monochromatic extraordinary beam centred about a slightlydifferent wavelength pass through and fall on a third mirror 28 thatreflects the beams onto a second prism 30. The second prism furtherdisperses the light and further separates the ordinary and extraordinarybeams, which are reflected via a fourth mirror 32 towards a slit 34. Theslit selects only one of the polarised states and defines the finalband-pass of the light. The prisms 24, 30 are of a Wollaston prismarrangement, and effectively double the separation of ordinary andextraordinary polarised beams compared to a single polarising Rochondesign, enabling twice the bandwidth to be selected. The monochromatoris arranged to maximise the light output.

The wavelength of light leaving the monochromator may be varied throughcontrol means which adjust the prisms in the monochromator. The beam oflight leaving the polarising monochromator for the present applicationideally comprises a series of ultra-violet wavelengths from the range180 nm and 260 nm, though of course the particular range may be chosento suit a particular application.

Referring to FIG. 3, the linearly polarised, monochromatic light 21emerging from the polarising monochromator exit slit 34 is focused bylenses 36, 38 onto a photo-elastic modulator (PEM), which converts athigh frequency the linearly polarised light into alternately left- andright-circularly polarised light 22. The alternately polarised light 22irradiates a sample placed in sample chamber 42.

Light of a particular wavelength may be absorbed by the sample, and inthe case of a molecule containing one or more chiral chromophores, suchas a protein, the absorption may be different for left- andright-circularly polarised light.

The sample is a contained in a suitable cell, which includes athermocouple and peltier device by which means the temperature of thecell is precisely and rapidly controlled.

A detector 46 placed after the sample detects how much left- andright-circularly polarised light is transmitted through the sample ateach wavelength, from which the difference in their absorption, i.e. thecircular dichroism, can be determined. The set of data gives a ΔAsurface, that is, a series of spectra as a function of temperature. Thedetector uses an avalanche photodiode detector in order to maximise thesignal to noise ratio.

As light is directed on to the sample and readings at differentwavelengths are taken, the sample temperature is continuously increased.A typical is heating regime may start at 4° C. and be raised at 1° C.per minute until the temperature reaches 95° C. In this way, a set ofdata is generated that gives a CD surface, where each point on thesurface is characterised by a value of CD corresponding to a precisewavelength and a precise temperature.

Referring to FIG. 4, the CD surface is projected onto the CD-wavelengthplane, with each CD spectrum, taken at intervals of 1° C., representedby a single line. The CD spectrum 40, measured at the beginning of theexperiment, has a shape that is typical of a well-folded protein of thetype under investigation. This is a positive indicator that the proteinis biologically active. As the temperature increases, there is littlechange between 4° C. and 40° C.; between approximately 40° C. and 60°C., the secondary structure changes significantly, as shown by theprogression of CD spectra indicated by arrows 42. Between approximately60° C. and 75° C., a second change in secondary structure occurs, asshown by the progression of CD spectra indicated by arrows 44. A furtherchange in the secondary structure of the protein takes place between 75°C. and 90° C. as shown by the progression of the CD spectra indicated bythe arrows 46.

Referring to FIG. 5, the CD surface is projected onto the CD-temperatureplane with each trace representing a CD-temperature profile at a givenwavelength. Two transitions between secondary structures can be seenclearly, having mid-points around 50° C. and 65° C., and possibly athird transition having a mid-point above 75° C.

The absorbance of the sample can be derived accurately and in real-timefrom the CD data. Referring to FIG. 6, the absorbance surface isprojected onto the absorbance-temperature plane with each tracerepresenting an absorbance-temperature profile at a given wavelength. Atwavelengths is where there is no chromophore and thus no possible trueabsorbance, e.g. trace 48, a change in the apparent absorbancecommencing at about 60° C. can be seen nonetheless. The change is due tolight scattering and absorbance is used as a proxy to monitor it. Lightis scattered by particles formed during aggregation. The aggregatingparticles reach such a size that they eventually precipitate and thiscan be seen in trace 48 and others as the absorbance profile decreasesfrom approximately 73° C. onwards.

The analysis of the data may be completely automated but typically isdone in a number of steps. Using singular value decomposition or similartechniques, the principal components in the data can be identified. Theprincipal components define the number of states present and thereforean appropriate model for the data can be identified. For example, atwo-state reversible transition can be modelled using the appropriatethermodynamic equations for a reversible two-state system. Usingnon-linear least-squares or similar techniques, the model can be refinedto give a best fit to the data. The model is used to calculate themid-point temperature and enthalpy for each transition, the spectra ofthe initial, final and any intermediate states (shown for the presentexample in FIG. 7), and the concentration profiles s of the states as afunction of temperature (shown for the present example in FIG. 8).

One can also calculate the transition surface for the three independentspecies together as a function of temperature wavelength and CDdifference (FIG. 9) which gives a three dimensional surface. Theresidual surface (i.e. the data which remains after the calculatedeffect of the three independent species have been subtracted from theoriginal data), may also be plotted in this way as shown in FIG. 10,which in this example seems to show a non-random fourth species.

With the optical arrangement in the monochromator and the detectionsystem described herein, it is possible to analyse many samples a day.The presentation of samples may be automated.

The same principle may be used when analysing a sample using othertechniques, such as measuring the fluorescence. Further, rather thanvarying the temperature with the optical quantity being measured,another independent variable, such as pH or sample concentration, couldbe changed whilst the optical quantity is being measured.

1. A spectrometer for analysing material, comprising: a light source;monochromator for selecting a range of wavelengths from the light sourceand emitting them as monochromatic light; a chamber for locating asample; a focusing means for focusing the monochromatic light onto asample in the chamber; a detector for measuring the monochromatic lightafter it has interacted with the sample; wherein an independentlyvariable parameter is varied between two values v1 and v2; and whereinthe detector measures the monochromatic light across a range ofwavelengths when the independent variable has a value or values betweenv1 and v1+Δv, where Δv is much smaller than the interval between v1 andv2.
 2. A spectrometer according to claim 1, including a polarising meansthat polarises the light into separate right and left circularlypolarised light.
 3. A spectrometer according to claim 1, wherein thedetector is an avalanche photodiode detector.
 4. A spectrometeraccording to claim 1, wherein the property of the sample which is variedis temperature.
 5. A spectrometer according to claim 1, wherein theproperty of the sample which is varied is pH.
 6. A spectrometeraccording to claim 1, wherein there is included software that acceptsthe data from the detector to determine values of the parameters whichare varied at which a transition in the sample occurs.
 7. A spectrometeraccording to claim 4, including software that accepts the data from thedetector to determine value of the enthalpy or other thermodynamicproperty of the sample that occurs during a transition.
 8. Aspectrometer for analysing material comprising; a light source; amonochromator for selecting a range of wavelengths from the light sourceand emitting them as monochromatic light; a chamber for locating asample; a focusing means for focusing the monochromatic light onto asample in the chamber; and a detector for measuring the monochromaticlight after it has interacted with the sample; wherein the detector isan avalanche photodiode detector.
 9. A spectrometer according to claim8, wherein a property of the sample is varied between two values v1 andv2, and wherein the detector measures the monochromatic light across arange of wavelengths when the sample has a value or values between v1and v1+Δv, where Δv is much smaller than the interval between v1 and v2.10. A spectrometer according to claim 8, wherein there is included apolarising means that polarises the light into separate right and leftcircularly polarised light.
 11. A spectrometer according to claim 8,wherein the property of the sample which is varied is temperature.
 12. Aspectrometer according to claim 8, wherein the property of the samplewhich is varied is pH.
 13. A spectrometer according to claim 8, whereinthere is included software that accepts the data from the detector todetermine value of the parameters which are varied at which a transitionin the sample occurs.
 14. A spectrometer according to claim 8, whereinthere is included software that accepts the data from the detector todetermine value of the enthalpy or other thermodynamic property of thesample that occurs during a transition.